Regulation of osteogenesis in bone marrow-derived mesenchymal stem cells via histone deacetylase 1 and 2 co-cultured with human gingival fibroblasts and periodontal ligament cells.

OBJECTIVE: This study aimed to determine the regulatory mechanism of bone marrow-derived mesenchymal stem cell (BM-MSC) differentiation mediated by humoral factors derived from human periodontal ligament (HPL) cells and human gingival fibroblasts (HGFs). We analyzed histone deacetylase (HDAC) expression and activity involved in BM-MSC differentiation and determined their regulatory effects in co-cultures of BM-MSCs with HPL cells or HGFs. BACKGROUND: BM-MSCs can differentiate into various cell types and can, thus, be used in periodontal regenerative therapy. However, the mechanism underlying their differentiation remains unclear. Transplanted BM-MSCs are affected by periodontal cells via direct contact or secretion of humoral factors. Therefore, their activity is regulated by humoral factors derived from HPL cells or HGFs. METHODS: BM-MSCs were indirectly co-cultured with HPL cells or HGFs under osteogenic or growth conditions and then analyzed for osteogenesis, HDAC1 and HDAC2 expression and activity, and histone H3 acetylation. BM-MSCs were treated with trichostatin A, or their HDAC1 or HDAC2 expression was silenced or overexpressed during osteogenesis. Subsequently, they were evaluated for osteogenesis or the effects of HDAC activity. RESULTS: BM-MSCs co-cultured with HPL cells or HGFs showed suppressed osteogenesis, HDAC1 and HDAC2 expression, and HDAC phosphorylation; however, histone H3 acetylation was enhanced. Trichostatin A treatment remarkably suppressed osteogenesis, decreasing HDAC expression and enhancing histone H3 acetylation. HDAC1 and HDAC2 silencing negatively regulated osteogenesis in BM-MSCs to the same extent as that achieved by indirect co-culture with HPL cells or HGFs. Conversely, their overexpression positively regulated osteogenesis in BM-MSCs. CONCLUSION: The suppressive effects of HPL cells and HGFs on BM-MSC osteogenesis were regulated by HDAC expression and histone H3 acetylation to a greater extent than that mediated by HDAC activity. Therefore, regulation of HDAC expression has prospects in clinical applications for effective periodontal regeneration, mainly, bone regeneration.

Healing effects of monomer and dimer resveratrol in a mouse periodontitis model.

BACKGROUND: The antioxidant and anti-inflammatory effects of resveratrol have been reported previously. Particularly, monomeric trans-resveratrol has been demonstrated to produce positive effects in various pathological processes. We reported previously that resveratrol dimer-rich melinjo extract, among others, caused bone healing, decreased local oxidative damage, and activated antioxidants nuclear factor erythroid 2-related factor 2 (Nrf2) pathways in a mouse model of experimentally induced periodontitis (EP). This study aimed to compare the bone-healing effects of the resveratrol monomer to the resveratrol dimer (gnetin C found in melinjo seed extract) in a model of EP and investigate the involvement of Nrf2 for effects of either form of resveratrol. METHODS: EP was induced experimentally in mice by placement of a 9 - 0 silk ligature around the left second molar. Mice received 10 mg/kg of either resveratrol monomer or dimer intraperitoneally on day 15 after induction of EP. The bone level around the ligated teeth was measured over time, and levels of proinflammatory cytokines and oxidative stress were measured in the periodontal tissues around the ligated teeth. RESULTS: Resveratrol dimer induced greater periodontal bone healing as compared to that related to use of the resveratrol monomer. It appears that healing of periodontal bone in either group was likely related to master regulation of antioxidant nuclear factor erythroid 2-related factor 2 (Nrf2) significantly. Downregulation of IL-1ß, a proinflammatory cytokine was also demonstrated in the resveratrol dimer group. CONCLUSION: Our results showed that administration of resveratrol in either dimer form or the monomeric form reduced periodontal bone loss with greater inhibition of bone loss being demonstrated in the dimer group as compared to the monomer group and that these effects were related in all likelihood to decreased oxidative stress and hence reduction in local inflammation.

Oral infectious bacteria in dental plaque and saliva as risk factors in patients with esophageal cancer.

BACKGROUND: High levels of periodontopathic bacteria as well as Streptococcus anginosus were detected in cancer tissue from patients with esophageal cancer. An association between oral infectious bacteria and esophageal cancer has been reported. METHODS: Characteristics of the oral microbiota and periodontal conditions were studied as clinicopathologic factors in patients with esophageal cancer. The study included 61 patients with esophageal cancer and 62 matched individuals without any cancers. Samples of subgingival dental plaque and unstimulated saliva were collected to evaluate the prevalence and abundance of the following oral bacteria using a real-time polymerase chain reaction assay: Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Treponema denticola, and S. anginosus. RESULTS: In the cancer group, the prevalence of all bacteria, with the exception of F. nucleatum, in dental plaque; the prevalence of A. actinomycetemcomitans in saliva; the abundance of all bacteria, with the exception of F. nucleatum and P. intermedia, in dental plaque; and the abundance of A. actinomycetemcomitans and S. anginosus in saliva were significantly higher. Furthermore, a logistic regression analysis suggested that the prevalence of T. forsythia and S. anginosus in dental plaque and of A. actinomycetemcomitans in saliva, as well as a drinking habit, were associated with a high risk of esophageal cancer, with a high odds ratio. CONCLUSIONS: The current findings have potential implications for the early diagnosis of esophageal cancer.

Identification of regulatory mRNA and microRNA for differentiation into cementoblasts and periodontal ligament cells.

OBJECTIVE: Periodontitis causes periodontal tissue destruction and results in physiological tooth dysfunction. Therefore, periodontal regeneration is ideal therapy for periodontitis. Mesenchymal stem cells (MSCs) are useful for periodontal regenerative therapy as they can differentiate into periodontal cells; however, the underlying regulatory mechanism is unclear. In this study, we attempted to identify regulatory genes involved in periodontal cell differentiation and clarify the differentiation mechanism for effective periodontal regenerative therapy. BACKGROUND: The cementum and periodontal ligament play important roles in physiological tooth function. Therefore, cementum and periodontal ligament regeneration are critical for periodontal regenerative therapy. Mesenchymal stem cell transplantation can be a common periodontal regenerative therapy because these cells have multipotency and self-renewal ability, which induces new cementum or periodontal ligament formation. Moreover, MSCs can differentiate into cementoblasts. Cementoblast- or periodontal ligament cell-specific proteins have been reported; however, it is unclear how these proteins are regulated. MicroRNA (miRNA) can also act as a key regulator of MSC function. Therefore, in this study, we identified regulatory genes involved in cementoblast or periodontal cell differentiation and commitment. METHODS: Human MSCs (hMSCs), cementoblasts (HCEM), and periodontal ligament cells (HPL cells) were cultured, and mRNA or miRNA expression was evaluated. Additionally, cementoblast-specific genes were overexpressed or suppressed in hMSCs and their expression levels were investigated. RESULTS: HCEM and HPL cells expressed characteristic genes, of which we focused on ets variant 1 (ETV1), miR-628-5p, and miR-383 because ETV1 is a differentiation-related transcription factor, miR-628-5p was the second-highest expressed gene in HCEM and lowest expressed gene in HPL cells, and miR-383 was the highest expressed gene in HCEM. miR-628-5p and miR-383 overexpression in hMSCs regulated ETV1 mRNA expression, and miR-383 overexpression downregulated miR-628-5p expression. Moreover, miR-383 suppression decreased miR-383 expression and enhanced ETV1 mRNA expression, but miR-383 suppression also decreased miR-628-5p. Furthermore, silencing of ETV1 expression in hMSCs regulated miR-628-5p and miR-383 expression. Concerning periodontal cell commitment, miR-628-5p, miR-383, and ETV1 regulated the expression of HCEM- or HPL cell-related genes by adjusting the expression of these miRNAs. CONCLUSION: HCEM and HPL cells show characteristic mRNA and miRNA profiles. In particular, these cells have specific miR-383, miR-628-5p, and ETV1 expression patterns, and these genes interact with each other. Therefore, miR-383, miR-628-5p, and ETV1 are key genes involved in cementogenesis or HPL cell differentiation.

Periodontal ligament cells regulate osteogenesis via miR-299-5p in mesenchymal stem cells.

BACKGROUND: The periodontal ligament contains periodontal ligament cells, which is a heterogeneous cell population, and includes progenitor cells that can differentiate into osteoblasts/cementoblasts. Mesenchymal stem cells (MSCs) can differentiate into various cells and can be used for periodontal regenerative therapy. Therefore, transplanted MSCs can be affected by humoral factors from periodontal ligament cells via the transcription factors or microRNAs (miRNAs) of MSCs. In addition, periostin (POSTN) is secreted from HPL cells and can regulate periodontal regeneration and homeostasis. To clarify the regulatory mechanism of humoral factors from periodontal ligament cells, we attempted to identify key genes, specifically microRNAs, involved in this process. METHODS: Human MSCs (hMSCs) were indirectly co-cultured with human periodontal ligament cells (HPL cells) and then evaluated for osteogenesis, undifferentiated MSCs markers, and miRNA profiles. Furthermore, hMSCs were indirectly co-cultured with HPL cells in the presence of anti-POSTN monoclonal antibody (anti-POSTN Ab) to block the effect of POSTN from HPL cells, and then evaluated for osteogenesis or undifferentiated MSC markers. Moreover, hMSCs showed alterations in miRNA expression or cultured with HPL were challenged with POSTN during osteogenesis, and cells were evaluated for osteogenesis or undifferentiated MSC markers. RESULTS: hMSCs co-cultured with HPL cells showed suppressed osteogenesis and characteristic expression of SOX11, an undifferentiated MSC marker, as well as miR-299-5p. Overexpression of miR-299-5p regulated osteogenesis and SOX11 expression as observed with indirect co-culture with HPL cells. Furthermore, MSCs co-cultured with HPL cells were recovered from the suppression of osteogenesis and SOX11 mRNA expression by anti-POSTN Ab. However, POSTN induced miR-299-5p and SOX11 expression, and enhanced osteogenesis. CONCLUSION: Humoral factors from HPL cells suppressed osteogenesis in hMSCs. The suppressive effect was mediated by miR-299-5p and SOX11 in hMSCs.

A comparative questionnaire study of patient complaint levels between magnetostrictive ultrasonic scaler (Cavitron® ) and piezoelectric ultrasonic scalers.

OBJECTIVES: Ultrasonic scalers often cause an uncomfortable feeling to patients during the procedure. This study was conducted to compare patient complaint levels between magnetostrictive (M-USSC) and piezoelectric ultrasonic scalers (P-USSC) during supragingival scaling. METHODS: This study enrolled 82 subjects who received supportive periodontal therapy for at least 2 years. At each recall visit, probing pocket depth (PPD), bleeding on probing (BOP) and O'Leary plaque control record (O'PCR) were recorded. Then, supragingival scaling was performed using P-USSC (Varios or Petit Piezo) at the first visit and M-USSC (Cavitron) at the second visit. After each treatment, a questionnaire survey was performed using the Wong-Baker Faces Pain Scale for six items, which included the typical complaints occurring during ultrasonic scaling. RESULTS: The scores for all the six items related to patient complaints were greater for P-USSC than for M-USSC (p < 0.001). Patient complaints such as discomfort, pain, sound, vibration, hyperesthesia and length of treatment time were ameliorated in 74%, 65%, 80%, 67%, 57% and 53% of subjects using M-USSC, respectively. On the other hand, only <5% of subjects showed deterioration in terms of each complaint. CONCLUSION: This study suggested that M-USSC causes fewer patient complaints during supragingival scaling than P-USSC. M-USSC may improve patient motivation and compliance and may contribute towards achieving successful treatment outcomes. However, this result could differ depending on the shape of the tip and the insert and treatment site. Further research will be required under various conditions.

Regulation of osteogenesis via miR-101-3p in mesenchymal stem cells by human gingival fibroblasts.

INTRODUCTION: Mesenchymal stem cells (MSCs) can differentiate into various types of cells and can thus be used for periodontal regenerative therapy. However, the mechanism of differentiation is still unclear. Transplanted MSCs are, via their transcription factors or microRNAs (miRNAs), affected by periodontal cells with direct contact or secretion of humoral factors. Therefore, transplanted MSCs are regulated by humoral factors from human gingival fibroblasts (HGF). Moreover, insulin-like growth factor (IGF)-1 is secreted from HGF and regulates periodontal regeneration. To clarify the regulatory mechanism for MSC differentiation by humoral factors from HGF, we identified key genes, specifically miRNAs, involved in this process, and determined their function in MSC differentiation. MATERIALS AND METHODS: Mesenchymal stem cells were indirectly co-cultured with HGF in osteogenic or growth conditions and then evaluated for osteogenesis, undifferentiated MSC markers, and characteristic miRNAs. MSCs had their miRNA expression levels adjusted or were challenged with IGF-1 during osteogenesis, or both of which were performed, and then, MSCs were evaluated for osteogenesis or undifferentiated MSC markers. RESULTS: Mesenchymal stem cells co-cultured with HGF showed suppression of osteogenesis and characteristic expression of ETV1, an undifferentiated MSC marker, as well as miR-101-3p. Over-expression of miR-101-3p regulated osteogenesis and ETV1 expression as well as indirect co-culture with HGF. IGF-1 induced miR-101-3p and ETV1 expression. However, IGF-1 did not suppress osteogenesis. CONCLUSIONS: Humoral factors from HGF suppressed osteogenesis in MSCs. The effect was regulated by miRNAs and undifferentiated MSC markers. miR-101-3p and ETV1 were the key factors and were regulated by IGF-1.

Japanese subgingival microbiota in health vs disease and their roles in predicted functions associated with periodontitis.

The present study aimed to identify and compare the microbial signatures between periodontally healthy and periodontitis subjects using 454 sequences of 16S rRNA genes. Subgingival plaque samples were collected from ten periodontally healthy subjects and ten matched chronic periodontitis patients. Bacterial DNA was extracted and next-generation sequencing of 16S rRNA genes was performed. The microbial composition differed between healthy subjects and periodontitis patients at all phylogenetic levels. Particularly, 16 species, including Lautropia mirabilis and Neisseria subflava predominated in healthy subjects, whereas nine species, including Porphyromonas gingivalis and Filifactor alocis predominated in periodontitis. UniFrac, a principal coordinate and network analysis, confirmed distinct community profiles in healthy subjects and periodontitis patients. Using predicted function profiling, pathways involved in phenylpropanoid, GPI-anchor biosynthesis, and metabolism of alanine, arginine, aspartate, butanoate, cyanoamino acid, fatty acid, glutamate, methane, proline, and vitamin B6 were significantly over-represented in periodontitis patients. These results highlight the oral microbiota alterations in microbial composition in periodontitis and suggest the genes and metabolic pathways associated with health and periodontitis. Our findings help to further elucidate microbial composition and interactions in health and periodontitis.

Deep sequencing reveals specific bacterial signatures in the subgingival microbiota of healthy subjects.

OBJECTIVES: This study aimed to define the comprehensive bacterial flora of the healthy oral cavity by identifying and comparing bacterial species in different subgingival sites using 454 sequencing of 16S rRNA genes. MATERIALS AND METHODS: Subgingival plaque samples were taken from six target teeth (central incisor, first premolar, and first molar in both the maxilla and mandible) of 10 periodontally healthy patients. Bacterial DNA was extracted and next-generation sequencing of 16S rRNA genes was performed. RESULTS: Bacterial composition in phylum level was similar for all sites within the same individual irrespective of tooth location. Unweighted UniFrac distance values of microbiome also showed that average distance was significantly larger between subjects than between tooth locations of the same subjects. CONCLUSIONS: The present results clarify the lack of effect of tooth location in the healthy subgingival microbiota. Results may suggest that any subgingival site can demonstrate similar subject-specific microbiota. CLINICAL RELEVANCE: This investigation offers a better understanding of the uniqueness of the oral microbiome. The present study will facilitate sampling in future subgingival microbiological studies.

[The effect of routine professional oral care on oral mucositis in hematologic chemotherapy patients].

BACKGROUND: According to the literature, 40%of all oral complications associated with chemotherapy are due to oral mucositis. Moreover, such complications increase the difficulties associated with oral intake, leading to deterioration of the patient's nutritional condition and increasing the risk of systemic infection. Therefore, oral mucositis prevention and proper treatment are very important. PATIENTS AND METHODS: The conditions of intra-oral cavities and effects of oral care in patients with hematological malignancies were retrospectively evaluated by dental hygienists from April 2008 to March 2011. RESULTS: Eleven of 28 patients(39.3%)who received routine professional oral care developed oral mucositis. In many such patients, intra-oral cavity deterioration, evidenced by a coated tongue and Candida infection, was observed. Although 25 of 28 patients with hematologic malignancies received specific oral mucositis care after chemotherapy initiation, those receiving continuous oral care subsequently made a full recovery. CONCLUSIONS: These results suggest that early and continuous professional oral health care may play an important role in the effective chemotherapy of patients with hematologic malignancies.

Amelotin Promotes Mineralization and Adhesion in Collagen-Based Systems.

Introduction: Periodontitis is characterized by the destruction of tooth-supporting tissues including the alveolar bone. Barrier membranes are used in dentistry for tissue regenerative therapy. Nevertheless, conventional membranes have issues related to membrane stability and direct induction of bone mineralization. Amelotin (AMTN), an enamel matrix protein, regulates hydroxyapatite crystal nucleation and growth. To apply an AMTN membrane in clinical practice, we investigated the mineralizing and adhesive effects of recombinant human (rh) AMTN in vitro using a collagen-based system. Methods: Collagen hydrogel incorporated with rhAMTN (AMTN gel) and rhAMTN-coated dentin slices were prepared. AMTN gel was then applied on a commercial membrane (AMTN membrane). Samples were incubated for up to 24 h in mineralization buffer, and the structures were observed. The peak adhesive tensile strength between the dentin and AMTN membrane was measured. Using an enzyme-linked immunosorbent assay, the release kinetics of rhAMTN from the membrane were investigated. Results: The AMTN gel resulted in the formation of hydroxyapatite deposits both onto and within the collagen matrix. Furthermore, coating the dentin surface with rhAMTN promoted the precipitation of mineral deposits on the surface. Interestingly, site-specific mineralization was observed in the AMTN membrane. Only 1% of rhAMTN was released from the membrane. Hence, the AMTN membrane adhered to the dentin surface with more than twofold greater tensile strength than that detected for a rhAMTN-free barrier membrane. Conclusions: RhAMTN can accelerate mineralization and adhesion in collagen-based systems. Furthermore, the AMTN membrane could inform the optimal design of calcified tissue regenerative materials. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-022-00722-2.

Overexpression of PD-L1 in gingival basal keratinocytes reduces periodontal inflammation in a ligature-induced periodontitis model.

BACKGROUND: The immune checkpoint programmed cell death 1 (PD-1): PD-1 ligand 1 (PD-L1) pathway plays a crucial role in maintaining immune tolerance and preventing tissue damages by excessive immune responses. PD-L1 is physiologically expressed and upregulated in keratinocytes (KCs) in the oral cavity. We here investigated the contribution of PD-L1 that was overexpressed in gingival basal KCs in a ligature-induced periodontitis model. METHODS: Wild-type (WT) BALB/c and K14/PD-L1 transgenic (tg) mice, in which PD-L1 was overexpressed in basal KCs under control of the keratin 14 promoter, were used. To induce periodontitis, a 9-0 silk ligature was placed around the upper right second molar, and lipopolysaccharide from Porphyromonas gingivalis was applied on the suture. Gingival tissues were collected on day 7, after which histological analyses were performed, including by hematoxylin and eosin and tartrate-resistant acid phosphate staining (TRAP) and quantitative PCR for proinflammatory cytokines and bone metabolism-related genes. Alveolar bone loss at 7 weeks after ligature placement was assessed by micro-computed tomography analysis. RESULTS: PD-L1 was overexpressed in the basal KCs of all gingival epithelia in K14/PD-L1tg mice. Early ligature-induced periodontal inflammation, as assessed based on histological changes, elevation of proinflammatory cytokine (IL-1ß, IL-6, TNF-α) expression, periodontal ligament degeneration, and osteoclastogenesis as assessed by Rankl and Opg expression and TRAP+ cells, was markedly impaired in K14/PD-L1tg mice. Alveolar bone resorption at a late time point was also clearly minimized in K14/PD-L1tg mice. CONCLUSION: Overexpression of PD-L1 in gingival basal keratinocytes in K14/PD-L1tg mice reduces periodontal inflammation and alveolar bone resorption in a ligature-induced periodontitis model.

Effect of Amelotin on Bone Growth in the Murine Calvarial Defect Model.

Amelotin (AMTN) is a protein that is expressed during the maturation of dental enamel and has important role in enamel hydroxyapatite mineralization. However, it is not well understood whether AMTN has a strong mineral-promoting ability in bone. In this study, the effect of AMTN on bone healing was investigated using mice calvarial defect model in vivo, and the expression of bone marker genes and cell proliferation were investigated to clarify the role of AMTN in bone mineralization using mouse osteogenic cells (MC3T3-E1) in vitro. Collagen membranes, with or without recombinant human (rh) AMTN, were applied to calvarial defects created on the parietal bones of C57BL/6N mice. Microcomputed tomography and histological observation revealed that the defect largely filled with mineralized tissue by the rhAMTN-containing membrane in eight weeks. Moreover, CD31 positive cells were observed in the newly formed mineralized tissue and around the rhAMTN-containing membrane. In the presence of rhAMTN, the expression of the Spp1 gene in MC3T3-E1 cells significantly increased within ten days in an osteoinductive medium. Moreover, rhAMTN significantly enhanced MC3T3-E1 cell proliferation. These findings indicate that AMTN positively influences bone repair by promoting hydroxyapatite mineralization.

Resveratrol derivative-rich melinjo seed extract induces healing in a murine model of established periodontitis.

BACKGROUND: Products of internal defense systems, like pro-inflammatory cytokines, reactive oxygen species, and leukocytes, are released which attack periodontal bacteria in periodontitis, but at the same time, lead to tissue destruction as well. We hypothesize that resveratrol derivative-rich melinjo seed extract (MSE), an edible plant extract that has antioxidant properties, should promote healing of periodontal bone loss and modulating immune-inflammatory systems that leads periodontal tissue destruction. METHODS: We used an experimentally induced periodontitis (EP) model in mice. Ligatures were placed first for development of EP (15 days). MSE was intraperitoneally administrated (0.001% (w/w)) to reverse bone loss that had already occurred in established EP and mice were then sacrificed (day 17, 20 and 22). RESULTS: Morphometric outcomes revealed lower bone-loss in the MSE groups compared to control. Immunohistochemistry assays demonstrated lower oxidative stress in MSE groups. MSE also inhibited M-CSF/sRANKL mediated osteoclast formation and down-regulated osteoclast activity. CONCLUSIONS: Treatment with MSE in EP actually caused healing of bone, and these effects are probably related to decreases in local oxidative damage and osteoclast activity. Given MSE's positive effects on osteodifferentiation as well, these findings suggest that MSE could be a useful therapeutic agent for the management of periodontitis.

Wear characteristics of trimethylolpropane trimethacrylate filler-containing resins for the full crown restoration of primary molars.

Although the demand for aesthetic restoration of primary molars has increased, the full-crown restorations using resin and the details of the wear characteristics of trimethylolpropane trimethacrylate (TMPT) filler containing resins for primary molars are not well understood. This study was conducted to determine whether new light-cured composite resin (Fantasista) and 4-META/MMATBB resin (Bondfill SB) are appropriate for full crown restoration of primary molars by evaluating their wear characteristics. Both resins products contain TMPT filler. The properties of the resins were evaluated through in vitro impacting-sliding wear tests; the wear properties of the opposing enamel specimens used in the tests were also studied. The properties of the resins were compared with those of Litefill, MetafilC, and Clearfil FII, which had been evaluated previously. Fantasista exhibited simple shape of wear that was suggestive of a higher wear resistance than that of Litefill. Fantasista caused the least damage to the antagonistic primary enamel.